Cell Biology Chapter 9
Compare and contrast microtubule assembly and intermediate filament assembly.
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Contrast the apparent roles of kinesin and cytoplasmic dynein in axonal transport
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Describe three functions of microtubules.
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how is each protofilament assembled
Each protofilament is assembled from dimeric building blocks consisting of one α-tubulin and one β-tubulin subunit. The two types of globular tubulin subunits have a similar three-dimensional structure and fit tightly together as shown in Figure 9.3c. The tubulin dimers are organized in a linear array along the length of each protofilament, as shown in Figure 9.3d. Because each assembly unit contains two nonidentical components (a heterodimer), the protofilament is asymmetric, with an α-tubulin at one end and a β-tubulin at the other end. All of the protofilaments of a microtubule have the same polarity. Consequently, the entire polymer has polarity.
We will restrict the present discussion to classes I-IV, which are found in the construction of cytoplasmic filaments, and consider type V IFs (the lamins), which are present as part of the inner lining of the nuclear envelope, in
IFs radiate through the cytoplasm of a wide variety of animal cells and are often interconnected to other cytoskeletal filaments by thin, wispy cross-bridges (FIGURE 9.33). In many cells, these cross-bridges consist of an elongated dimeric protein called plectin that can exist in numerous isoforms. Each plectin molecule has a binding site for an intermediate filament at one end and, depending on the isoform, a binding site for another intermediate filament, microfilament, or microtubule at the other end.
All microtubules of the axoneme have the same polarity
Their plus ends are at the tip of the projection and their minus ends at the base. Each peripheral doublet consists of one complete microtubule, the A tubule, and one incomplete microtubule, the B tubule, the latter containing 10 or 11 subunits rather than the usual 13.
intraflagellar transport (IFT
When cilia and flagella assemble, all growth is restricted to their distal tips, and this requires a way for proteins made inside the cell to get out to the tip where assembly is taking place. Biologists had been watching the flagella of living cells for over a hundred years, but it wasn't until 1993 that Joel Rosenbaum and colleagues at Yale University observed the movement of particles in the space between the peripheral doublets and the surrounding plasma membrane and named this movement intraflagellar transport (IFT
defects in both anterograde and retrograde tramsportt
are linked to several neurological disease including amyotrophic lateral sclerosis (ALS), also known as lou gehrig's disease
he polypeptide subunits of IFs can be divided into five major classes
based on the type of cell in which they are found (Table 9.2) as well as biochemical, genetic, and immunologic criteria.
other structures, including endocytic vesicles that form at the axon terminals aren carry regulatory factors from target cells, move
in the opposite, or retrograde direction from the synapse toward the cell body
Keratin filaments constitute the primary structural proteins of epithelial cells
including epidermal cells, liver hepatocytes, and pancreatic acinar cells
The basic building block of IF assembly
is thought to be a rodlike tetramer formed by two dimers that become aligned side by side in a staggered fashion with their N- and C-termini pointing in opposite (antiparallel) directions
motor proteins can be grouped into three broad superfamilies
kinesins, dyneins, and myosins. Kinesins and dyneins move along microtubules, whereas myosins move along actin filaments.
plus and minus ends of microtubules
one end of microtubule is plus end and is terminated by a row of beta tubules subunits -the opposite end is the minus end and is terminated by a row of alpha tubules subunits -the structural polarity of microtubules is an important factor in the growth of these structures and their ability to participate in directed mechanical activities
kinesin
one member of a superfamily of related proteins, called KRPs (kinesin-related proteins). KRPs are classified into 14 different families (kinesin-1 to kinesin-14). In the following discussion the term kinesin will refer only to members of the kinesin-1 family.
how do motor proteins move
unidirectionally along their cytoskeletal track in a stepwise manner from one binding site to the next - The steps in the chemical cycle include the binding of an ATP molecule to the motor, the hydrolysis of the ATP, the release of the products (ADP and Pi) from the motor, and the binding of a new molecule of ATP. The binding and hydrolysis of a single ATP molecule at the catalytic site is used to drive a power stroke that moves the motor a precise number of nanometers along its track
Certain animal cells, including mouse oocytes, lack centrosomes entirely
yet they are still capable of forming complex microtubular structures, such as the meiotic spindle (as discussed in Chapter 14). Human patients with genetic defects in centrosome-associated proteins display microcephaly, a reduction in brain size, presumably because the neuronal proliferation and migration are particularly sensitive to reductions in centrosome function.
Actin
- Actin is also involved in intracellular motile processes, such as the movement of vesicles, phagocytosis, and cytokinesis. -Actin also plays an important role in determining the shapes of cells and can provide structural support for various types of cellular projections
structure of microtubule
-hollow, tubular structures, assembled from the protein tubules. They are found in the cytoskeleton, the mitotic spindle, centrioles, and the core of cilia and flagella -function to support cell and movement of materials between the cell body and axon terminals of a neuron
centrosome
-the microtubules of the cytoskeleton are typically nucleated by this -a complex structure that contains two barrel-shaped centrioles surrounded by amorphous, electron-dense pericentriolar material (or PCM)
A body of evidence suggests at least two well-studied roles for cytoplasmic dynein:
1) As a force-generating agent in positioning the spindle and moving chromosomes during mitosis (discussed in Chapter 14). 2) As a minus end-directed microtubular motor with a role in positioning the centrosome and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm.
If a typical plant cell is followed from one mitotic division to the next, four distinct arrays of microtubules appear, one after another
1)During most of interphase, the microtubules of a plant cell are distributed widely throughout the cortex, as depicted in Figure 9.15, stage 1. A search for γ-tubulin shows this nucleation factor to be localized along the lengths of the cortical microtubules, suggesting that new microtubules might form directly on the surface of existing microtubules. This idea is supported by studies of tubulin incorporation in living cells (FIGURE 9.16a) and by in vitro assays (Figure 9.16b) that show newly formed microtubules branching at an angle off the sides of preexisting microtubules. Once formed, the daughter microtubules are likely severed from the parent microtubule and incorporated into the parallel bundles that encircle the cell (Figures 9.6a, 9.15). 2)As the cell approaches mitosis, the microtubules disappear from most of the cortex, leaving only a single transverse band, called the preprophase band, that encircles the cell like a belt (Figure 9.15, stage 2). The preprophase band marks the site of the future division plane. 3)As the cell progresses into mitosis, the preprophase band is lost and microtubules reappear in the form of the mitotic spindle (Figure 9.15, stage 3). 4)After the chromosomes have been separated, the mitotic spindle disappears and is replaced by a bundle of microtubules called the phragmoplast (Figure 9.15, stage 4), which plays a role in the formation of the cell wall that separates the two daughter cells (see Figure 14.38).
These dramatic changes in the spatial organization of microtubules are thought to be accomplished by a combination of two separate mechanisms
(1) the rearrangement of existing microtubules and (2) the disassembly of existing microtubules and reassembly of new ones in different regions of the cell. In the latter case, the microtubules that make up the preprophase band are formed from the same subunits that a few minutes earlier were part of the cortical array or, before that, the phragmoplast.
dynein
-responsible for movement of cilia and flagella
Contrast the sensory and motile functions of cilia and flagella. Describe how failures of these different ciliary functions contribute to human diseases.
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What are five functions of the cytoskeleton?
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how can disassembly be induced
Disassembly can be induced by cold temperature; hydrostatic pressure; elevated Ca2+ concentration; and a variety of chemicals, including colchicine, vinblastine, vincristine, and nocodazole.
Polymerization of a microtubule can push on an attached object, whereas depolymerization of a microtubule can pull on an attached object
For instance, this type of pulling force plays a major role in the segregation of chromosomes during cell division
The dynamic character of the microtubular cytoskeleton inside a cell can be revealed by expressing
GFP-tagged tubulin in living cells or injecting tubulin with chemical tags that allow it to be visualized in fixed cells (FIGURE 9.20). If individual microtubules are observed under a fluorescence microscope, they appear to grow slowly for a period of time and then shrink rapidly and unexpectedly, as illustrated by the microtubule being followed in FIGURE 9.21. Because the switch from growth to shrinkage (an event termed catastrophe) occurs with high frequency in vivo, most microtubules disappear from the cell in a matter of minutes and are replaced by new microtubules that grow out from the centrosome
the distribution of cytoplasmic microtubules in a cell helps determine the shape of a cell
In cultured animal cells, the microtubules extend in a radial array outward from the area around the nucleus, giving these cells their round, flattened shape (FIGURE 9.5). In contrast, the microtubules of columnar epithelial cells are typically oriented with their long axis parallel to the long axis of the cell (Figure 9.1a). This configuration suggests that microtubules help support the cell's elongated shape.
how do plant cells rely on actin
In fact, plant cells rely primarily on actin, rather than microtubules, to serve as tracks for the long-distance transport of cytoplasmic vesicles and organelles. This bias toward actin-based motility reflects the rather restricted distribution of microtubules in many plant cells
intermediate filaments
Intermediate filaments are strong, flexible, ropelike fibers that provide mechanical strength to cells that are subjected to physical stress, including neurons, muscle cells, and the epithelial cells that line the body's cavities. Unlike actin filaments and microtubules, IFs are a chemically heterogeneous group of structures that, in humans, are encoded by approximately 70 different genes.
cells contain a diverse array of proteins (called +TIPs) that bind to the dynamic plus ends of microtubules
Some of these +TIPs regulate the rate of the microtubule's growth or shrinkage or the frequency of interconversion between the two phases. Other +TIPs mediate the attachment of the plus end of the microtubule to a specific cellular structure, such as the kinetochore of a mitotic chromosome during cell division or the actin cytoskeleton of the cortex during vesicle transport.
These IFs, or neurofilaments, as they are called
The cytoplasm of neurons contains loosely packed bundles of intermediate filaments whose long axes are oriented parallel to that of the axon (see Figure 9.7b). These IFs, or neurofilaments, as they are called, are composed of three distinct proteins: NF-L, NF-H, and NF-M, all of the type IV group o
In most cells, as in axons, microtubules are aligned with their plus ends pointed away from the center of the cell
Therefore, members of the kinesin superfamily tend to move vesicles and organelles (e.g., peroxisomes and mitochondria) in an outward direction toward the cell's plasma membrane.
Why has such a costly polymerization pathway evolved?
To answer this question, it is useful to consider the effect of GTP hydrolysis on microtubule structure. When a microtubule is growing, the plus end is seen under the electron microscope as an open sheet to which GTP-dimers are added (step 1, FIGURE 9.19a, 9.19b). During times of rapid microtubule growth, tubulin dimers are added more rapidly than their GTP can be hydrolyzed. The presence of a cap of tubulin-GTP dimers at the plus ends of the protofilaments is thought to favor the addition of more subunits and the growth of the microtubule. However, microtubules with open ends, as in step 1, Figure 9.19a, are thought to undergo a spontaneous reaction that leads to closure of the tube (steps 2 and 3). In this model, tube closure is accompanied by the hydrolysis of the bound GTP, which generates subunits that contain GDP-bound tubulin. GDP-tubulin subunits have a different conformation from their GTP-tubulin precursors and are less suited to fit into a straight protofilament. The resulting mechanical strain destabilizes the microtubule. The strain energy is released as the protofilaments curl outward from the plus end of the tubule and undergo catastrophic depolymerization (Figure 9.19a, step 4; 9.19c). Thus, it would appear that GTP hydrolysis is a fundamental component of the dynamic quality of microtubules. The strain energy stored in a microtubule as a result of GTP hydrolysis makes the microtubule inherently unstable and—in the absence of other stabilizing factors such as MAPs—capable of disassembling soon after its formation. Microtubules can shrink remarkably fast, especially in vivo, which allows a cell to disassemble its microtubular cytoskeleton very rapidly.
cytoplasmic dynein
a huge protein (molecular mass of approximately 1.5 million daltons) composed of two identical heavy chains and a variety of intermediate and light chains (Figure 9.11a). Each dynein heavy chain consists of a large globular head with an elongated projection (stalk). The dynein head, which is an order of magnitude larger than a kinesin head, acts as a force-generating engine. Each stalk contains the all-important microtubule-binding site situated at its tip. The longer projection, known as the stem (or tail), binds the intermediate and light chains, whose functions are not well defined.
When studied in vitro, the assembly of microtubules from αβ-tubulin dimers occurs in two distinct phases
a slow phase of nucleation, in which a small portion of the microtubule is initially formed, and a much more rapid phase of elongation. Unlike the case in vitro, nucleation of microtubules takes place rapidly inside a cell, where it occurs in association with a variety of specialized structures called microtubule-organizing centers (or MTOCs). The best studied MTOC is the centrosome.
each kinesis-1 molecule
a tetramer constructed from two identical heavy chains and two identical light chains (FIGURE 9.9a). A kinesin molecule has several parts, including a pair of globular heads that bind a microtubule and act as ATP-hydrolyzing, force-generating "engines." Each head (or motor domain) is connected to a neck, a rodlike stalk, and a fan-shaped tail that binds cargo to be hauled (Figure 9.9a). The motor portions of all KRPs have related amino acid sequences, reflecting their common evolutionary ancestry and their similar role in moving along microtubules.
Although IF polypeptides have diverse amino acid sequences
all share a similar structural organization that allows them to form similar-looking filaments. Most notably, the polypeptides of IFs all contain a central, rod-shaped, α-helical domain of similar length and homologous amino acid sequence. This long fibrous domain makes the subunits of intermediate filaments very different from the globular tubulin and actin subunits of microtubules and actin filaments.
structures nad materials traveling form the cell body toward teh terminals of a neuron are said to move in an
anterograde direction
The moving particles seen in IFT
are complexes of 20 proteins, known as IFT proteins, which assemble into a protein complex called the IFT particle. These IFT particles then line up to form linear arrays called IFT trains, and the trains are moved out to the tip of the cilium by a plus-end directed kinesin motor, kinesin-2. IFT trains are then brought back to the cell body by a cytoplasmic dynein complex. The IFT particles carry cargo proteins such as tubulin, required for building cilia, out to the tip for assembly, and inhibition of IFT prevents assembly of cilia and flagella. Mutations in genes that encode IFT proteins have been important tools in testing the function of cilia in development and physiology because they result in complete loss of cilia.
microtubules
long, hollow, unbranched tubes composed of subunits of the protein tubules -the tracks over which peroxisomes are transported in mammalian cells -the force-generating apparatus that moves cells from one place to another -cilia and flagella
in plant cells microtubules play a role in
maintaining cell shape through their influence on the formation of the cell wall
In the presence of ATP, actin monomers
polymerize to form a flexible, helical filament.
Give some examples that reinforce the suggestion that intermediate filaments are important primarily in tissue-specific functions rather than in basic activities that are common to all cells.
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What is the centrosome? What component of the centrosome nucleates microtubules and how does it dictate the number of protofilaments?
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What is the role of GTP in the assembly of microtubules? What is meant by the term dynamic instability? What role does it play in cellular activity?
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not all cilia are motile
Almost all cells of the human body contain a single nonmotile cilium (called a primary cilium) that is thought to have a sensory function, monitoring the mechanical and chemical properties of extracellular fluids
Early in vitro studies established that GTP is required for microtubule assembly.
Assembly of tubulin dimers requires that a GTP molecule be bound to the β-tubulin subunit.2 As it turns out, β-tubulin is not only a structural protein, it is also an enzyme, a GTPase. GTP hydrolysis is not required for the actual incorporation of the dimer onto the end of a microtubule. Rather, the GTP is hydrolyzed to GDP shortly after the dimer is incorporated into a microtubule, and the resulting GDP remains bound to the assembled polymer. After a dimer is released from a microtubule during disassembly and enters the soluble pool, the GDP is replaced by a new GTP. This nucleotide exchange "recharges" the dimer, allowing it to serve once again as a building block for polymerization. Because it includes GTP hydrolysis, assembly of a microtubule is not an inexpensive cellular activity.
Dynamic instability provides a mechanism by which the plus ends of microtubules can rapidly explore the cytoplasm for appropriate sites of attachment
Attachment temporarily stabilizes microtubules and allows the cell to build the complex cytoskeletal networks discussed in this chapter. Dynamic instability also allows cells to respond rapidly to changing conditions that require remodeling of the microtubular cytoskeleton. One of the most dramatic examples of such remodeling occurs at mitosis when the microtubules of the cytoskeleton are disassembled and remodeled into a bipolar mitotic spindle. This reorganization is associated with a marked change in microtubule stability; microtubules in interphase cells have half-lives that are 5 to 10 times longer than microtubules in mitotic cells. Unlike the microtubules of the mitotic spindle or cytoskeleton, the microtubules of the organelles to be discussed below lack dynamic activity and, instead, are highly stable.
transmembrane protein whose cytoplasmic side is physically attached to cortical microtubules through a protein called
CSI1 (Cellulose Synthase Interactive Protein 1). As a result, the cellulose microfibrils of the cell wall are assembled in an orientation that is parallel to the underlying microtubules of the cortex (FIGURE 9.6). The orientation of these cellulose microfibrils plays an important role in determining the growth characteristics of the cell and thus its shape.
Centrioles are cylindrical structures about 0.2 µm in diameter and typically about twice as long.
Centrioles contain nine evenly spaced blades, each of which contain three microtubules, designated the A, B, and C tubules. Only the A tubule is a complete microtubule (Figure 9.12a,b). The nine A tubules are connected to a central hub with nine spokes called the cartwheel. The nine-fold symmetry of the centriole results from the structure of the SAS-6 protein, which self-assembles into a ninefold symmetric disc that forms the core of the cartwheel.
basal body
Centrosomes are not the only MTOCs in cells. The outer microtubules in a cilium or flagellum are generated as outgrowths from the microtubules in a structure called a basal body, which resides at the base of the cilium or flagellum -basal bodies are identical in structure to centrioles, and can turn into centrioles and vice versa
Timothy Mitchison and Marc Kirschner of the University of California, San Francisco, reported on the properties of individual microtubules and proposed that microtubule behavior in vivo could be explained by a phenomenon they termed dynamic instability
Dynamic instability explains the observation (1) that growing and shrinking microtubules can coexist in the same region of a cell and (2) that a given microtubule can switch back and forth unpredictably (stochastically) between growing and shortening phases,
Although all microtubules appear quite similar morphologically, there are marked differences in their stability
Microtubules of the mitotic spindle or the cytoskeleton are extremely labile, that is, sensitive to disassembly. Microtubules of mature neurons are much less labile, and those of centrioles, cilia, and flagella are highly stable. These differences in microtubule stability are determined by microtubule interacting proteins including MAPs (Figure 9.4) which stabilize microtubules, proteins known as +TIPs, which bind to the plus-end of growing microtubules, and an enzyme called katanin, named after the samurai sword, that severs microtubules into shorter pieces.
microtubule-associated proteins (or MAPs)
Microtubules prepared from living tissue typically contain additional proteins, called microtubule-associated proteins (or MAPs). MAPs comprise a heterogeneous collection of proteins. The first MAPs to be identified are referred to as "classical MAPs" and typically have one domain that attaches to the side of a microtubule and another domain that projects outward as a tail from the microtubule's surface.
Because the dimers point in opposite directions, the tetramer itself lacks polarity.
Recent studies of the self-assembly of IFs in vitro suggest that eight tetramers associate with one another in a side-by-side (lateral) arrangement to form a filament that is one unit in length (about 60 nm) (step 4). Subsequent growth of the polymer is accomplished as these unit lengths of filaments associate with one another in an end-to-end fashion to form the highly elongated intermediate filament (step 5). None of these assembly steps is thought to require the direct involvement of either ATP or GTP. Because the tetrameric building blocks lack polarity, so too does the assembled filament, which is another important feature that distinguishes IFs from other cytoskeletal elements.
in humans, called epidermolysis bullosa simplex (EBS).3 Subsequent analysis of EBS patients has shown that they carry mutations in the gene that encodes the homologous K14 polypeptide (or the K5 polypeptide, which forms dimers with K14). These studies confirm the role of IFs in imparting mechanical strength to cells situated in epithelial layers.
Similarly, knockout mice that fail to produce the desmin polypeptide exhibit serious cardiac and skeletal muscle abnormalities. Desmin plays a key structural role in maintaining the alignment of the myofibrils of a muscle cell, and the absence of these IFs makes the cells extremely fragile. An inherited human disease, named desmin-related myopathy, is caused by mutations in the gene that encodes desmin. Persons with this disorder suffer from skeletal muscle weakness, cardiac arrhythmias, and eventual congestive heart failure. Not all IF polypeptides have such essential functions. For example, mice that lack the vimentin gene, which is expressed in fibroblasts, macrophages, and white blood cells, show relatively minor abnormalities, even though the affected cells lack cytoplasmic IFs. It is evident from these studies that IFs have tissue-specific functions, which are more important in some cells than in others.
he nexin link component of the N-DRC forms an elastic linkage that connects adjacent doublets (Figure 9.26b). These nexin bridges play an important role in ciliary/flagellar movement by limiting the extent that adjacent doublets can slide over one another. The resistance to sliding provided by the nexin bridges causes the axoneme to bend in response to the sliding force.
Sliding on one side of the axoneme alternates with sliding on the other side so that a part of the cilium or flagellum bends first in one direction and then in the opposite direction (FIGURE 9.32). This requires that, at any given time, the dynein arms on one side of the axoneme are active, while those on the other side are inactive. As a result of this difference in dynein activity, the doublets on the inner side of the bend (at the top and bottom of Figure 9.32) extend beyond those on the opposite side of the axoneme. A major open question in the cilia field is what mechanism coordinates the motion of thousands of dynein arms during the ciliary beating cycle. The central pair contributes to this coordination by signaling to the dynein arms through the radial spokes; however, motility is still possible in mutant flagella that lack the central pair, arguing that the dynein arm activities can self-organize in some manner which remains be to determined.
When labeled keratin subunits are injected into cultured skin cells, they are rapidly incorporated into existing IFs
Surprisingly, the subunits are not incorporated at the ends of the filament, as might have been expected by analogy with microtubule and actin filament assembly, but rather into the filament's interior (FIGURE 9.35a). The results depicted in Figure 9.35 might reflect the exchange of unit lengths of filament directly into an existing IF network (as shown in step 6, Figure 9.34a). Unlike the other two major cytoskeletal elements, assembly and disassembly of IFs are controlled primarily by phosphorylation and dephosphorylation of the subunits. For example, phosphorylation of vimentin filaments by protein kinase A leads to their disassembly.
The basic structure of the axoneme
The central tubules were seen to be enclosed by the central sheath, which is connected to the A tubules of the peripheral doublets by a set of radial spokes. The doublets are connected to one another by an interdoublet bridge composed of an elastic protein-based linkage called the nexin link. The nexin link is now known to be part of a biochemical complex containing proteins that regulate dynein activity, called the nexin-dynein regulatory complex or N-DRC. Of particular importance was the observation that a pair of "arms"—an inner arm and an outer arm—project from the A tubule (Figure 9.26a,b). These arms are composed of axonemal dyneins, protein complexes related to cytoplasmic dynein that are responsible for generating the bending motion in cilia and flagella. A longitudinal section, that is, one cut through the axoneme parallel to its long axis, reveals the continuous nature of the microtubules and the discontinuous nature of the other elements
Unlike the polypeptides of other IFs, NF-H and NF-M have sidearms that project outward from the neurofilament
These sidearms are thought to maintain the proper spacing between the parallel neurofilaments of the axon (see Figure 9.7b). In the early stages of differentiation when the axon is growing toward a target cell, it contains very few neurofilaments but large numbers of supporting microtubules. Once the nerve cell has become fully extended, it becomes filled with neurofilaments that provide support as the axon increases dramatically in diameter. Aggregation of NFs is seen in several human neurodegenerative disorders, including ALS and Parkinson's disease. These NF aggregates may block axonal transport, leading to the death of neurons.
Regardless of their diverse appearance, all MTOCs play similar roles in all cells
They control the number of microtubules, their polarity, the number of protofilaments that make up their walls, and the time and location of their assembly. In addition, all MTOCs share a common protein component—a type of tubulin called γ-tubulin
γ-tubulin
Unlike the α- and β-tubulins, which make up about 2.5 percent of the protein of a nonneural cell, γ-tubulin constitutes only about 0.005 percent of the cell's total protein. Fluorescent antibodies to γ-tubulin stain all types of MTOCs, including the pericentriolar material of centrosomes (Figure 9.14a), suggesting that γ-tubulin is a critical component in microtubule nucleation. This conclusion is supported by other studies. For example, mutations in γ tubulin lead to reduced microtubule nucleation at MTOCs.
In its power stroke, the cilium is maintained in a rigid state
as it pushes against the surrounding medium. In its recovery stroke, the cilium becomes flexible, offering little resistance to the medium. Cilia tend to occur in large numbers on a cell's surface, and their beating activity is usually coordinated
The terms actin filament, F-actin, and microfilament
basically synonyms for this type of filament. Depending on the type of cell and the activity in which it is engaged, actin filaments can be organized into ordered arrays, highly branched networks, or tightly anchored bundles.
how does kinesis move
by a hand over hand mechanism -two heads alternate taking the leading and lagging position without an accompanying rotation of the stalk and cargo at every step -the movement is highly processive meaning that the motor protein tends to move along an individual microtubule for a considerable distances over 1µm without falling off
the wall of a microtubule
composed of globular proteins arranged in longitudinal rows, termed protofilemnts, that are aligned parallel to the long axis of the tubule -when viewed in cross section, microtubules are seen to consist of 13 protofilemtns aligned side b side in a circular pattern within the wall
primary cilia
contain receptors and channels that allow them to serve as sensory antennas. Specialized sensory cilia include cilia that sense fluid flow in the kidney, olfactory cilia in the nose that sense smell, and the outer segments of rod and cone cells in the retina, which are in fact highly specialized primary cilia.
The core of the cilium, called the axoneme
contains an array of microtubules that runs longitudinally through the entire organelle. With rare exceptions, the axoneme of a motile cilium or flagellum consists of nine peripheral doublet microtubules surrounding a central pair of single microtubules. This same microtubular structure, which is known as the 9 + 2 array, is seen in axonemes from protists to mammals and serves as another of the many reminders that all living eukaryotes have evolved from a common ancestor.
the motor proteins of a cell
convert chemical energy (ATP) into mechanical energy, which is used to generate force, as occurs when a muscle cell contracts or to move cellular cargo
In such cases, the basal bodies do not form by the typical process of centriole duplication, but rather assemble in large amorphous structures called
deuterosomes
what do MAPS due
generally increase stability of microtubule and promote their assembly
motor proteins
generate forces required to move objects within a cell
cilia and flagell
hairlike, sometimes motile organelles that project from the surface of a variety of eukaryotic cells. Many cells in the human body contain a single non-motile cilium
An abnormally high level of phosphorylation of one particular MAP, called tau
has been implicated in the development of several fatal neurodegenerative disorders, including Alzheimer's disease (Section 2.13). The brain cells of people with these diseases contain strange, tangled filaments (called neurofibrillary tangles) consisting of tau molecules that are excessively phosphorylated and unable to bind to microtubules. The neurofibrillary filaments have been proposed to contribute to the death of nerve cells, but whether these filaments actually help cause neuronal degeneration or are just a consequence of neuronal death is still a controversial point.
Because centrosomes are sites of microtubule nucleation, the microtubules of the cytoskeleton are all polarized the same way
he minus end is associated with the centrosome, and the plus (or growing) end is situated at the opposite tip (Figure 9.13b). The fraction of microtubules that remain associated with the centrosome is highly variable from one cell type to another. The centrosome of a nonpolarized cell (e.g., a fibroblast) is typically situated near the center of the cell and tends to remain associated with the minus ends of a large number of microtubules (as in Figure 9.12e). In contrast, many of the microtubules in a polarized epithelial cell are anchored by their minus ends at dispersed sites near the apical end of the cell as their plus ends extend toward the cell's basal surface (Figure 9.1). Similarly, the axon of a nerve cell contains large numbers of microtubules that have no association with the centrosome, which is located in the neuron's cell body. Many of these axonal microtubules are thought to form in conjunction with the centrosome, but are then severed from the MTOC and transported into the axon by motor proteins.
One small family (called kinesin-14)
including the widely studied Ncd protein of Drosophila, moves in the opposite direction, that is, toward the minus end of the microtubular track. One might expect that the heads of plus end-directed and minus end-directed KRPs would have a different structure because they contain the catalytic motor domains. But the heads of the two proteins are virtually indistinguishable. Instead, differences in directional movement have been traced to differences in the adjacent neck regions of the two proteins. When the head of a minus end-directed Ncd molecule is joined to the neck and stalk portions of a kinesin-1 molecule, the hybrid protein moves toward the plus end of the track. Thus, even though the hybrid has a catalytic domain that normally would move toward the minus end of a microtubule, as long as it is joined to the neck of a plus end motor, it moves in the plus direction.
The central fibrous domain
is flanked on each side by globular domains of variable size and sequence (step 1, FIGURE 9.34). Two such polypeptides spontaneously interact as their α-helical rods wrap around each other to form a ropelike dimer approximately 45 nm in length (step 2). Because the two polypeptides are aligned parallel to one another in the same orientation, the dimer has polarity, with one end defined by the C-termini of the polypeptides and the opposite end by their N-termini.
In the intact axoneme, the stem of each dynein molecule (with its associated intermediate and light chains)
is tightly anchored to the outer surface of the A tubule, with the globular heads and stalks projecting toward the B tubule of the neighboring doublet
Although actin filaments can generate forces on their own
most processes involving actin require the activity of motor proteins, specifically those of the myosin superfamily.
A third small family (kinesin-13)
of kinesin-like proteins are incapable of movement. The KRPs of this group bind to either end of a microtubule and bring about its depolymerization rather than moving along its length. These proteins are often referred to as microtubule depolymerases
In vitro motility assays indicate that cytoplasmic dynein moves processively along a microtubule toward the polymer's minus end
opposite that of most kinesins
If the basal body arises from a centriole that had already been present during the preceding cell division, the cilium that it makes is called the
primary cilium -usually non-motile
each of the three types of cytoskeletal filaments is a polymer of
protein subunits held together by weak, non covalent bonds
All of the monomers within an actin filament are pointed in the same direction
resulting in a polar filament with so-called "barbed" and "pointed" ends
types of cellular cargo transported by the proteins
ribonucleoprotein particles, vesicles, mitochondria, lysosomes, chromosomes, and other cytoskeletal filaments.
In some cells that make large numbers of cilia, the additional cilia are nucleated by new basal bodies that form after division, and hence are called
secondary cilia
actin filaments
solid, thinner structures, often organized into a branching network
the drug tacol
stops the dynamic activities of microtubules by a different mechanism -Taxol binds to the microtubule polymer, inhibiting its disassembly, thereby preventing the cell from assembling new microtubular structures as required. Many of these compounds, including taxol, are used in chemotherapy against cancer because they preferentially kill tumor cells.
According to this proposal, the dynein arms pictured in FIGURE 9.30 act as
swinging cross-bridges that generate the forces required for ciliary or flagellar movement.
Keratin-containing IFs radiate through the cytoplasm
tethered to the nuclear envelope in the center of the cell and anchored at the outer edge of the cell by connections to the cytoplasmic plaques of desmosomes and hemidesmosomes (Section 7.9). Figure 9.36a also depicts the interconnections between IFs and the cell's microtubules and actin filaments, which transforms these otherwise separate elements into an integrated cytoskeleton. Because of these various physical connections, the IF network is able to serve as a scaffold for organizing and maintaining cellular architecture and for absorbing mechanical stresses applied by the extracellular environment.
manufacturing center of a neuron
the cell body, a rounded portion of the cell
Microtubule stability is also regulated by posttranslational modifications to the tubulin subunits such as
the covalent attachment of multiple glutamates onto the C-terminus of tubulin. Living cells can be subjected to a variety of artificial treatments that lead to the disassembly of labile cytoskeletal microtubules without disrupting other cellular structures.
In step 1 of Figure 9.31, the dynein arms anchored along the A tubule of the lower doublet attach to binding sites on the B tubule of the upper doublet. In step 2
the dynein molecules undergo a conformational change, or power stroke, that causes the lower doublet to slide toward the basal end of the upper doublet. This conformational change in a dynein heavy chain is depicted in Figure 9.31b-d. In step 3 of Figure 9.31, the dynein arms have detached from the B tubule of the upper doublet. In step 4, the arms have reattached to the upper doublet so that another cycle can begin. (An electron micrograph of an axoneme showing the dynein arms from one doublet attached to the adjacent doublet is shown in Figure 18.21.)
cytoskeleton
the eukaryotic skeletal system -composed of three well-defined filamentous structures - microtubules, actin filaments, and intermediate filaments- that tougher roark an elaborate interactive and dynamic network
intermediate filaments
tough, ropelike fibers composed of a variety of related proteins
Like kinesin-1, most KRPs move
toward the plus end of the microtubule to which they are bound
flagella
typically occur singly or in pairs and exhibit a variety of different beating patterns (waveforms), depending on the cell type. For example, the single-celled alga pictured in FIGURE 9.25a pulls itself forward by waving its two flagella in an asymmetric manner that resembles the breaststroke of a human swimmer
